The present invention relates to methods for detecting the presence or absence of methylated CpG islands within a genome utilizing a microarray based technology, Differential Methylation Hybridization (DMH). The invention is also used for identifying methylation patterns in a cell sample which may be indicative of a disease state. Also provided are methods for preparing nucleic acid fragments and nucleic acid probes to be used in said DMH methods.
Epigenetic events are heritable alterations in gene function which are mediated by factors other than changes in primary DNA sequence. DNA methylation is one of the most widely studied epigenetic mechanisms and numerous studies have been conducted to determine its role in oncogenesis. DNA methylation usually occurs at cytosines located 5xe2x80x2 of guanines, known as CpG dinucleotides, in the human genome. DNA (cytosine-5)-methyltransferase (DNA-MTase) catalyzes this reaction by adding a methyl group from S-adenosyl-L-methionine to the fifth carbon position of the cytosines. While DNA-MTase favors hemimethylated substrates for its normal maintenance activity in the cell, the enzyme also exhibits an ability to methylate CpG dinucleotides de novo. Most cytosines within the CpG dinucleotides are methylated in the human genome, but some remain unmethylated in specific CpG dinucleotide rich genomic regions, known as CpG islands. See Antequera, F. et al., Cell 62: 503-514 (1990).
Methylation of CpG islands is known to play a critical role in regulating gene expression. This effect is exerted via altering local chromatin structure and limiting the access of protein factors to initiate gene transcription. In normal cells, this epigenetic modification is associated with transcriptional silencing of imprinted genes, some repetitive elements and genes on the inactive X chromosome. See Li et al., Nature 366: 362-365 (1993); Singer-Sam, J. and Riggs A. D., (1993) In Jost, J. P., and Saluz, H. P. (eds), xe2x80x9cDNA Methylation: Molecular Biology and Biological Significance,xe2x80x9d p.358-384. In neoplastic cells, it has been observed that the normally unmethylated CpG islands can become aberrently methylated, or hypermethylated. See Jones, P. A., Cancer Res. 56: 2463-2467 (1996); Baylin et al., Advances in Cancer Research, In Vande Woude, G. F.: and Klein, G. (eds) 72: 141-196 (1997).
In addition to classic genetic mutations, hypermethylation of CpG islands is an alternative mechanism for inactivation of tumor suppressor genes and there is growing evidence that altered cytosine methylation patterns play important roles in cancer development. See e.g., Belinsky et al., 95 Proc. Natl. Acad. Sci. USA 11891-11896 (1998); Baylin et al., Advances in Cancer Research, In Vande Woude, G. F. and Klein, G. (eds.) 72: 141-196 (1997). The methylation patterns of DNA from cancer tumor cells are generally different than those of normal cells. See Laird et al., Hum. Mol. Genet. 3: 1487-1495 (1994). Tumor cell DNA is generally undermethylated relative to normal cell DNA, but selected regions of the tumor cell genome may be more methylated than the same regions of a normal cell genome. Hence, detection of altered methylation patterns in a tumor cell genome is an indication that the cell is cancerous.
Recently, the molecular mechanisms underlying CpG island hypermethylation in cancer have been explored and evidence suggests that increased DNA-MTase levels can contribute to tumorigenesis by promoting de novo methylation of CpG island sequences. See Vertino et al., Mol. Cell Biol., 16: 4555-4565 (1996); Wu et al., Cancer Res., 56: 616-622 (1996). For instance, if hypermethylation occurs in the CpG islands of genes related to growth-inhibitory activities, it may lead to associated transcriptional silencing and promote neoplastic cell proliferation. Further, recent data has shown:that dysregulation of p21, a cell cycle regulator that normally modulates DNA-MTase action may also promote de novo methylation. See Chuang et al., Science 277: 1996-2000 (1997). Studies have suggested that local cis-acting signals and trans-acting factors capable of preventing specific CpG islands from de novo methylation can be disrupted in tumor cells. See Brandeis, M. et al., Nature, 371: 435-438 (1995); Mummaneni, P. et al., J. Biol. Chem., 270: 788-792 (1995); Graff et al., J. Biol. Chem., 272: 22322-22329 (1997).
Presently, there is no direct evidence that disturbances of such local factors results in de novo methylation of specific CpG islands. Rather, de novo methylation is commonly thought to be a generalized phenomenon associated with a stochastic process in tumor cells possessing aberrant DNA-MTase activities. See Jones, P. A., Cancer Res., 56, 2463-2467 (1996); Pfeifer et al., Proc. Natl. Acad. Sci. USA, 87: 8252-8256. (1990). This random methylation process can occur at CpG dinucleotide sites located within the regulatory regions of tumor suppressor genes. The progressive silencing of their transcripts may provide tumor cells with a growth advantage, and the specific hypermethylated sites observed in particular cancer types could be the result of clonal selection during tumor development.
Thus, identification of genetic changes in tumorigenesis is a major focus in molecular cancer research. However, the differences in CpG island methylation patterns between normal and cancer cells remain poorly understood.
Traditionally, methylation analysis has been carried out by Southern hybridization which assesses a few methylation-sensitive restriction sites within CpG islands of known genes. More sensitive assays for mapping DNA methylation patterns such as bisulfite DNA sequencing and methylation-specific PCR, have allowed a detailed analysis of multiple CpG dinucleotides across a single CpG island of interest. Bisulfite DNA sequencing utilizes bisulfite-induced modification of genomic DNA under conditions whereby unmethylated cytosine is converted to uracil. The bisulfite-modified sequence is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues are amplified as thymine and only 5-methylcytosine residues are amplified as cytosine. The PCR products can be sequenced or can be cloned and sequenced to provide methylation maps of single DNA molecules. See Frommer, M. et al., Proc. Natl. Acad. Sci. 89: 1827-1831 (1992).
Similarly, methylation-specific PCR, another widely used assay, can assess the methylation status of CpG dinucleotide sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails the initial modification of DNA by sodium bisulfite or another comparable agent thus converting all unmethylated, but not methylated, cytosines to uracil. Subsequent amplification with primers specific for methylated DNA results in the amplification of DNA consisting of methylated CpG dinucleotides. See U.S. Pat. No. 5,786,146; Herman et al., Proc. Natl. Acad. Sci. USA 93: 9821-9826 (1996).
These approaches have yielded important information regarding the local methylation control of individual genes. However, current methods have been restricted to analyzing one gene at a time and have not been used to conduct a genome-wide study. As a further step toward a more comprehensive understanding of the underlying mechanisms, it is necessary to perform large-scale or a genome-wide analysis of methylation patterns of DNA in cancer cells.
Accordingly, a need presently exists for technology designed to detect methylation of DNA on a large scale, to identify previously uncharacterized CpG islands associated with gene silencing and to shed light on other, as yet unidentified factors governing aberrant methylation of CpG island loci. Each cancer type may have its own unique methylation pattern that defines its growth rate, tendency to spread, and responsiveness to therapies. By examining a large number of loci in a series of cancers, global methylation profiles can be constructed. Cataloging these molecular patterns could lead to early detection, more accurate diagnosis, and development of better treatment therapies of cancer.
Accordingly, among the objects of the present invention may be noted the provision of a novel DNA array-based method, differential methylation hybridization (DMH) to detect the presence or absence of hypermethylated nucleic acid sequences in a cell sample. DMH utilizes a set of CpG dinucleotide rich fragments prepared from tumor cells or normal cells to simultaneously screen numerous genomic nucleic acid fragments. The use of DMH provides an accurate and efficient method for the identification of DNA methylation patterns in cancer and thus, DMH has wide-ranging applications in clinical diagnosis and genetic typing of cancer.
An object of the present invention is to provide a process for detecting the presence or absence of methylation of a CpG dinucleotide rich region of a nucleic acid sequence within a genome. A nucleic acid sequence is digested with a enzyme which digests nucleic acid sequences into fragments in which CpG islands are preserved. These fragments containing the CpG islands are then digested with a methylation-sensitive enzyme resulting in a digestion product comprising methylated CpG island loci. The digestion product is amplified and labeled to form amplicons which are used to screen a plurality of nucleic acid fragments affixed to a solid support. The presence or absence of labeled amplicons bound to the plurality of nucleic acid fragments of the screening array is then determined.
It is another object of the present invention to provide a process for identifying methylation patterns in a cancer cell using amplicons generated from cancer and non-cancer cells to screen an array containing genomic fragments.
Another object of the present invention is to provide a screening array comprising a solid support and a plurality of CpG dinucleotide rich fragments affixed to the solid support. The CpG dinucleotide rich fragments are at least about 200 nucleotides in length and contain at least 50% guanine and cytosine.
Yet another object of the present invention is to provide a process for generating a screening array comprising a plurality of nucleic acid fragments containing expressed sequences which includes contacting a nucleic acid sequence with an enzyme which digests the nucleic acid sequences into fragments in which CpG islands are preserved; amplifying and screening the fragments to identify sequences which include expressed sequences and affixing the fragments containing expressed sequences to a solid support. It is another object of the present invention to provide a set of amplicons to be used to probe the nucleic acid fragments affixed on a solid support of the screening array. The amplicons are CpG dinucleotide rich fragments which are derived from digesting a nucleic acid sequence with a restriction enzyme which digests the sequence into fragments in which CpG dinucleotide fragments are preserved. The resulting digestion products are then amplified and used to probe nucleic acid fragments of the screening array.
Other objects and features of the present invention will be in part apparent and in part pointed out hereinafter.